Project Summary: Despite large improvements in breast cancer therapy, toxic side effects from chemotherapy are a major limitation; thus, it is necessary to develop novel therapeutic strategies that specifically target cancer cells while sparing healthy cells. One hallmark of cancer cells is the ?Warburg Effect,? a phenomenon where cancer cells exhibit accelerated glucose metabolism in the presence of oxygen. Hexokinase catalyzes the first committed step in glucose metabolism by phosphorylating glucose to glucose-6-phosphate and thereby trapping glucose in the cell to be used in various downstream pathways. Previous research has demonstrated that while normal mammary gland cells do not express the hexokinase 2 (HK2) isoform, it is highly overexpressed in breast cancer cells, which, in part, is responsible for the accelerated glucose utilization in primary tumors. HK2 deletion inhibits the tumorigenicity of cancer cells in vitro and in vivo. More importantly, systemic deletion of HK2 after tumor onset inhibits tumor development without any adverse physiological consequences in mouse models of cancer. Specifically, HK1 expression levels are sufficient for normal cellular function, but the cancer cells cannot overcome the loss of HK2. As a result, HK2 appears to be a good potential target for therapeutic treatment of primary breast cancer tumors. However, metastasis accounts for the high mortality rate in breast cancer, which makes it more important to elucidate the role HK2 has in breast cancer metastasis. In fact, preliminary results showing that systemic deletion of HK2 after tumor onset in a mouse model of breast cancer metastasis profoundly inhibits metastasis. The proposed research plan will help elucidate the specific mechanism(s) for HK2's role in breast cancer metastasis. The level of HK2 expression dramatically affects to the level of epithelial mesenchymal transition (EMT) protein expression, specifically the important transcription factor SNAIL. In addition, HK2 acts as scaffold to promote the phosphorylation and subsequent inactivation Glycogen Synthase Kinase 3 (GSK3?). SNAIL is phosphorylated by GSK3?, which it turn leads to the degradation of SNAIL. SNAIL is also regulated by O-GlcNAc modification, which helps to stabilize the protein level by suppressing its GSK3? phosphorylation-mediated degradation. Aim 1 will investigate the mechanism for HK2's role in EMT regulation. Furthermore, it is possible that breast cancer cells upregulate HK2 to combat the increased energetic stress that occurs during metastasis. Research has shown that anti-oxidants can enhance the metastatic potential in various cancers. The upregulation of HK2 can increase metabolite flux through the pentose phosphate pathway for NADPH, thereby allowing cells to recycle anti-oxidants, such as gluthathione. Aim 2 will determine HK2's role in intracellular levels of reactive oxygen species in relation to breast cancer metastasis. Overall, this study utilizes a comprehensive approach to elucidate the mechanism(s) for HK2's role in breast cancer metastasis and as a result will provide a new potential therapeutic target.